Figure 3. ROS-induced Jak and Src participate in EMT signaling, which are regulated by SOCS1.

HCT116 p53 +/+ cells were treated with H₂O₂ at 200 μM for different durations and analyzed for Jak and Src activation by Western blot A. HCT116 p53 +/+ cells were treated with H₂O₂ at 200 μM for 2 h with or without pretreatment of JAK-inhibitor AG490 B., or Src-inhibitor PP1 C. and changes in EMT markers and kinases were analyzed. HCT116 p53 +/+ sh & sh-SOCS1 cells were incubated in the absence or presence NAC, AG490 and PP1. Changes in EMT associated proteins or kinase were analyzed by Western blot D. To examine the molecular interaction of Src and SOCS1, HCT116 p53+/+ cells over-expressing SOCS1 were subject to co-immunoprecipitation assay. Cell lysates were incubated with rabbit monoclonal c-Src Ab or control rabbit IgG followed by protein A/G-agarose beads. The immunoprecipitated pellets were separated from the supernatant, which were resolved on SDS-PAGE along with total lysates, and subjected to immunoblotting to reveal SOCS1 and Src protein bands E.